Methods for increasing adiponectin

ABSTRACT

In certain aspects, the present invention provides compositions and methods for increasing adiponectin in a patient in need thereof by administering an antagonist of an ActRIIB signaling pathway. Examples of such antagonists include ActRIIB polypeptides, anti-ActRIIB antibodies, anti-activin A and/or B antibodies, anti-myostatin antibodies, anti-GDF3 antibodies, and anti-BMP7 antibodies. Also provided are methods for ameliorating one or more undesired effects of anti-androgen therapy, including muscle loss, bone loss, increased adiposity, and/or increased insulin resistance. A variety of disorders may be treated by causing an increase in circulating adiponectin concentrations.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/204,946 filed on Jan. 13, 2009. All the teachings of theabove-referenced application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Once thought to be merely an inert storage depot for excess energy,adipose tissue is now recognized as an active endocrine and paracrineorgan secreting multiple mediators, known as adipokines, thatparticipate in diverse metabolic processes. The polypeptide adipokineadiponectin is the most abundant known factor secreted by adipocytes andaccounts for approximately 0.01% of plasma protein. Whereas levels ofother adipokines increase with fat mass, adiponectin levels varyinversely with fat mass/obesity. Decreased adiponectin levels are alsoobserved in type 2 diabetes and cardiovascular disease. The strongcorrelation between low levels of circulating adiponectin, orhypoadiponectinemia, and risk factors for these major diseases mayderive partly from adiponectin's anti-inflammatory properties, whichcontrast with the proinflammatory character of other adipokines (Szmitkoet al., 2007, Am J Physiol Heart Circ Physiol 292:H1655-H1663). Thus,adiponectin appears to function as the protective adipokine,counterbalancing the potentially detrimental actions of these otheradipokines.

Considerable evidence has emerged linking hypoadiponectinemia withcardiovascular disease (Szmitko et al., supra). Adiponectin levels inpatients with coronary heart disease or cerebrovascular disease arelower than in healthy controls (Hotta et al., 2000, Arterioscler ThrombVasc Biol 20:1595-1599; Kumada et al., 2003, Arterioscler Thromb VascBiol 23:85-89; Pischon et al., 2004, JAMA 291:1730-1737) and varyinversely with the severity of disease. Hypoadiponectinemia isassociated with increased risk of cardiovascular disease even innonobese individuals (Im et al., 2006, Metabolism 55:1546-1550).Significantly, adiponectin inhibits development of atherosclerosis inanimal models (Okamoto et al., 2002, Circulation 106:2767-2770),providing evidence for a causal relationship between low adiponectinlevels and cardiovascular disease. Therefore, there is a need forActRIIB-derived agents and other inhibitors of ActRIIB signaling thatcan be used to treat or prevent hypoadiponectinemia.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides methods forincreasing adiponectin levels in patients in need thereof by usingantagonists of the ActRIIB signaling pathway. Patients in need of suchtherapy will typically exhibit low adiponectin, particularly in theserum. Such patients are considered to have a condition that is termedhypoadiponectinemia, Antagonists of the ActRIIB signaling pathway maybe, for example, soluble ActRIIB proteins (e.g., ActRIIB-Fc fusionproteins), antagonists that bind to ActRIIB or inhibit ActRIIBexpression, and antagonists that bind to or inhibit the expression ofligands that signal through ActRIIB and regulate adiponectin expressionand/or secretion. Such ligands may include myostatin, GDF3, activins(particularly activin A, activin B or activin AB), BMP7, BMP2 and BMP4.As demonstrated herein, ActRIIB-Fc fusion proteins can be used toincrease adiponectin gene expression and increase circulatingadiponectin levels in diverse mouse models.

In certain aspects, the disclosure provides methods for increasingadiponectin, or treating hypoadiponectinemia, by administering to apatient in need thereof an effective amount of an ActRIIB-relatedpolypeptide. An ActRIIB-related polypeptide may be an ActRIIBpolypeptide (e.g., an ActRIIB extracellular domain or portion thereof)that binds to an ActRIIB ligand such as GDF3, BMP2, BMP4, BMP7, GDF8,GDF11, activin A, activin B, activin AB or nodal. Optionally, theActRIIB polypeptide binds to an ActRIIB ligand with a Kd less than 10micromolar or less than 1 micromolar, 100, 10 or 1 nanomolar. A varietyof suitable ActRIIB polypeptides have been described in the followingpublished PCT patent applications, all of which are incorporated byreference herein: WO 00/43781, WO 04/039948, WO 06/012627, WO07/053,775, WO 08/097,541, and WO 08/109,167. Optionally, the ActRIIBpolypeptide inhibits ActRIIB signaling, such as intracellular signaltransduction events triggered by an ActRIIB ligand. A soluble ActRIIBpolypeptide for use in such a preparation may be any of those disclosedherein, such as a polypeptide having an amino acid sequence selectedfrom SEQ ID NOs: 1, 2, 5, 12, 23 and 26 or having an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an aminoacid sequence selected from SEQ ID NOs: δ 1, 2, 5, 12, 23 and 26. Asoluble ActRIIB polypeptide may include a functional fragment of anatural ActRIIB polypeptide, such as one comprising at least 10, 20 or30 amino acids of a sequence selected from SEQ ID NOs: 1, 2, 5, 12, 23and 26 or a sequence of SEQ ID NO: 1, lacking the C-terminal 1, 2, 3, 4,5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at theN-terminus. Optionally, polypeptides will comprise a truncation relativeto SEQ ID NO:1 of between 2 and 5 amino acids at the N-terminus and nomore than 3 amino acids at the C-terminus. Another polypeptide is thatpresented as SEQ ID NO:12. A soluble ActRIIB polypeptide may includeone, two, three, four, five or more alterations in the amino acidsequence (e.g., in the ligand-binding domain) relative to a naturallyoccurring ActRIIB polypeptide. The alteration in the amino acid sequencemay, for example, alter glycosylation of the polypeptide when producedin a mammalian, insect or other eukaryotic cell or alter proteolyticcleavage of the polypeptide relative to the naturally occurring ActRIIBpolypeptide. A soluble ActRIIB polypeptide may be a fusion protein thathas, as one domain, an ActRIIB polypeptide (e.g., a ligand-bindingdomain of an ActRIIB or a variant thereof) and one or more additionaldomains that provide a desirable property, such as improvedpharmacokinetics, easier purification, targeting to particular tissues,etc. For example, a domain of a fusion protein may enhance one or moreof in vivo stability, in vivo half life, uptake/administration, tissuelocalization or distribution, formation of protein complexes,multimerization of the fusion protein, and/or purification. A solubleActRIIB fusion protein may include an immunoglobulin constant domain,such as an Fc domain (wild-type or mutant) or a serum albumin. Incertain embodiments, an ActRIIB-Fc fusion comprises a relativelyunstructured linker positioned between the Fc domain and theextracellular ActRIIB domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIB (the “tail”), or it may be anartificial sequence of between 5 and 15, 20, 30, 50 or more amino acidsthat are relatively free of secondary structure. A linker may be rich inglycine and proline residues and may, for example, contain repeating ornon-repeating sequences of threonine/serine and/or glycines (e.g., TG₄,TG₃, SG₄, SG₃, G₄, G₃, G₂, G). A fusion protein may include apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIIBpolypeptide includes one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent. In general, it is preferable that anActRIIB protein be expressed in a mammalian cell line that mediatessuitably natural glycosylation of the ActRIIB protein so as to diminishthe likelihood of an unfavorable immune response in a patient. Human andCHO cell lines have been used successfully, and it is expected thatother common mammalian expression vectors will be useful.

In certain aspects, a compound disclosed herein may be formulated as apharmaceutical preparation for increasing adiponectin in a patient inneed thereof (e.g., the treatment of hypoadiponectinemia). Apharmaceutical preparation may also include one or more additionalcompounds such as a compound that is used to treat an ActRIIB-associateddisorder. Preferably, a pharmaceutical preparation is substantiallypyrogen free.

In certain aspects, the disclosure provides nucleic acids encoding asoluble ActRIIB polypeptide, which do not encode a complete ActRIIBpolypeptide. An isolated polynucleotide may comprise a coding sequencefor a soluble ActRIIB polypeptide, such as described above. For example,an isolated nucleic acid may include a sequence coding for anextracellular domain (e.g., ligand-binding domain) of an ActRIIBpolypeptide and a sequence that would code for part or all of thetransmembrane domain and/or the cytoplasmic domain of an ActRIIB, butfor a stop codon positioned within the transmembrane domain or thecytoplasmic domain, or positioned between the extracellular domain andthe transmembrane domain or cytoplasmic domain. For example, an isolatedpolynucleotide may comprise a full-length ActRIIB polynucleotidesequence such as SEQ ID NO: 4, or a partially truncated version, saidisolated polynucleotide further comprising a transcription terminationcodon at least six hundred nucleotides before the 3′-terminus orotherwise positioned such that translation of the polynucleotide givesrise to an extracellular domain optionally fused to a truncated portionof a full-length ActRIIB. Other suitable nucleic acids that encodeActRIIB polypeptides are shown as SEQ ID NO: 3, 4, 10 or 24. Nucleicacids disclosed herein may be operably linked to a promoter forexpression, and the disclosure provides cells transformed with suchrecombinant polynucleotides. Preferably the cell is a mammalian cellsuch as a CHO cell.

In certain aspects, the disclosure provides methods for making a solubleActRIIB polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 3, 4, 10 or 27) disclosed herein in asuitable cell, such as a Chinese hamster ovary (CHO) cell. Such a methodmay comprise: a) culturing a cell under conditions suitable forexpression of the soluble ActRIIB polypeptide, wherein said cell istransformed with a soluble ActRIIB expression construct; and b)recovering the soluble ActRIIB polypeptide so expressed. Soluble ActRIIBpolypeptides may be recovered as crude, partially purified or highlypurified fractions using any of the well known techniques for obtainingprotein from cell cultures.

In certain aspects, a compound described herein may be used in themanagement of a variety of forms of hypoadiponectinemia, includingpatients having low adiponectin and an associated condition (e.g.,atherosclerosis, ischemic stroke, impaired glucose tolerance, insulinresistance, diabetes type 2, hyperlipidemia, hypertriglyceridemia,obesity). As shown herein, ActRIIB polypeptides may be used to increaseadiponectin gene expression and/or circulating adiponectin levels whilealso having positive effects on body composition, specifically onmuscle, bone, and adipose tissue.

In certain aspects, the disclosure provides uses of a soluble ActRIIBpolypeptide for making a medicament for the treatment of a disorder orcondition as described herein.

In certain aspects, the disclosure provides methods for increasingadiponectin in a patient in need thereof (e.g., treatinghypoadiponectinemia), and such method may comprise administering aneffective amount of a compound selected from the group consisting of: apolypeptide comprising an amino acid sequence that is at least 90%, 93%,95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5, 23 or 26 and apolypeptide encoded by a nucleic acid that hybridizes under stringenthybridization conditions to a nucleic acid of SEQ ID NO: 3 or 24. Thepolypeptide may be a fusion protein comprising a heterologous portion.The polypeptide may be a dimer. The polypeptide may be fused to aconstant domain of an immunoglobulin. The polypeptide may be fused to anFc portion of an immunoglobulin, such as an IgG1, IgG2, IgG3 or IgG4.The polypeptide may comprise an amino acid sequence that is at least80%, 90%, 93%, 95%, 97%, 98%, 99% or 100% identical to the sequence ofamino acids 29-109, 29-128, 29-131, 29-134, 25-109, 25-128, 25-131,25-134 or 20-134 of SEQ ID NO:2. The polypeptide may comprise an aminoacid sequence that is at least 80%, 90%, 93%, 95%, 97%, 98%, 99% or 100%identical to the sequence of amino acids of SEQ ID NO: 1, 2, 5, 12, 23or 26. A patient to be treated with such a compound may be one having adisorder described herein, including, for example, hypoadiponectinemiaand associated conditions (e.g., atherosclerosis, ischemic stroke,impaired glucose tolerance, insulin resistance, diabetes type 2,hyperlipidemia, hypertriglyceridemia, or obesity).

In certain aspects, the disclosure provides methods for increasingadiponectin in a patient in need thereof (e.g., treatinghypoadiponectinemia), the method comprising administering an effectiveamount of a compound that inhibits the ActRIIB signaling pathway, eitherby targeting ActRIIB or a ligand that signals through ActRIIB. Examplesof such compounds include antagonists of ActRIIB; antagonists ofmyostatin; antagonists of activin A; antagonists of activin B;antagonists of BMP2; antagonists of BMP4 and antagonists of GDF3.Antagonists of each of the foregoing may be an antibody or other proteinthat specifically binds to and inhibits such target (e.g., an antibodysuch as a monoclonal antibody, or a propeptide in the case of myostatinand GDF3). Antagonists of the foregoing may also be a compound, such asa nucleic acid based compound (e.g., an antisense or RNAi nucleic acid)that inhibits the expression of ActRIIB or the ligand. A patient to betreated with such a compound may be one having a disorder describedherein, including, for example, low adiponectin level(hypoadiponectinemia), atherosclerosis, ischemic stroke, impairedglucose tolerance, insulin resistance, diabetes type 2, hyperlipidemia,hypertriglyceridemia, or obesity, and particularly any of the foregoingwherein the patient additionally exhibits low adiponectin levels.

In certain aspects, the disclosure provides methods for concurrentlyincreasing muscle, increasing bone, and increasing fat in a patient inneed thereof, the method comprising administering to the patient aneffective amount of an ActRIIB fusion protein, wherein the ActRIIBfusion protein comprises an amino acid sequence that is at least 90%,95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, 2, 5, 23 or 26.

In certain aspects, the disclosure provides methods for ameliorating oneor more undesired effects of anti-androgen therapy in a patient in needthereof, the method comprising administering to the patient an effectiveamount of an ActRIIB fusion protein, wherein the ActRIIB fusion proteincomprises an amino acid sequence that is at least 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 2, 5, 23 or 26. The undesired effectof anti-androgen therapy may be, for example, muscle loss, bone loss,increased adiposity, or increased insulin resistance, or combinations ofthe foregoing. In an exemplary embodiment, the undesired effect ofanti-androgen therapy is a combination of three or more of muscle loss,bone loss, increased adiposity and insulin resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a human ActRIIB soluble (extracellular) polypeptidesequence (SEQ ID NO: 1). The C-terminal “tail” is underlined.

FIG. 2 shows human ActRIIB precursor protein sequence (SEQ ID NO: 2).The signal peptide is underlined; the extracellular domain is in bold(also referred to as SEQ ID NO: 1); and the potential N-linkedglycosylation sites are boxed.

FIG. 3 shows a nucleic acid sequence encoding a human ActRIIB soluble(extracellular) polypeptide, designated as SEQ ID NO: 3.

FIG. 4 shows a nucleic acid sequence encoding human ActRIIB precursorprotein, designated as SEQ ID NO: 4.

FIG. 5 shows an alignment of human ActRIIA and ActRIIB with the residuesthat are deduced herein, based on composite analysis of multiple ActRIIBand ActRIIA crystal structures to directly contact ligand (the ligandbinding pocket) indicated with boxes.

FIG. 6 shows a multiple sequence alignment of various vertebrate ActRIIBproteins and human ActRIIA.

FIG. 7 shows the full amino acid sequence of ActRIIB(25-131)-hFc (SEQ IDNO: 23). The TPA leader (residues 1-22) and truncated ActRIIBextracellular domain (native residues 25-131) are each underlined.Highlighted is the glutamate revealed by sequencing to be the N-terminalamino acid of the mature fusion protein.

FIG. 8 shows a nucleotide sequence encoding ActRIIB(25-131)-hFc (SEQ IDNO: 24) (the coding strand is shown at top and the complement shown atbottom 3′-5′). Sequences encoding the TPA leader (nucleotides 1-66) andActRIIB extracellular domain (nucleotides 73-396) are underlined. Thecorresponding amino acid sequence for ActRIIB(25-131) is also shown.

FIG. 9 shows body weight vs. time in mice as a function of ORX andActRIIB(R64 20-134)-mFc treatment. Vehicle was Tris-buffered saline(TBS). Data shown are means (n=10 per group), and Day 71 means thatdiffer significantly (P<0.05, two-tailed unpaired t-test) are designatedby different letters. ActRIIB(R64 20-134)-mFc increased overall bodyweight gain under both ORX and gonad-intact conditions.

FIG. 10 shows lean body mass vs. time in mice as a function of ORX andActRIIB(R64 20-134)-mFc treatment. Lean body mass (total nonfat mass)was determined by NMR. Data shown are means (n=10 per group), and Day 71means that differ significantly (P<0.05, two-tailed unpaired t-test) aredesignated by different letters. Unlike ORX controls, ORX mice treatedwith ActRIIB(R64 20-134)-mFc gained lean body mass over the course ofthe experiment, finishing with values approximately 25% higher than inthe former group. A similar increase in lean body mass was also observedunder gonad-intact conditions for ActRIIB(R64 20-134)-mFc compared tovehicle.

FIG. 11 shows skeletal muscle mass in mice as a function of ORX andActRIIB(R64 20-134)-mFc treatment for 71 days. Pectoralis, rectusfemoris, and gastrocnemius muscles were surgically removed and weighedat study completion. Data shown are means±SEM (n=10 per group), andthose that differ significantly (P<0.05, two-tailed unpaired t-test) aredesignated by different letters. ActRIIB(R64 20-134)-mFc increased themass of all three muscles significantly under both ORX and gonad-intactconditions.

FIG. 12 shows whole-body bone area vs. time in mice as a function of ORXand ActRIIB(R64 20-134)-mFc treatment. Measurements were made by dualenergy X-ray absorptiometry (DEXA). Data shown are means (n=10 pergroup), and Day 47 means that differ significantly (P<0.05, two-tailedunpaired t-test) are designated by different letters. ActRIIB(R6420-134)-mFc prevented the progressive decrease in bone area observedunder ORX conditions and led to significantly increased bone area undergonad-intact conditions.

FIG. 13 shows whole-body bone mineral content vs. time in mice as afunction of ORX and ActRIIB(R64 20-134)-mFc treatment. Measurements weremade by dual energy X-ray absorptiometry (DEXA) analysis. Data shown aremeans (n=10 per group), and Day 47 means that differ significantly(P<0.05, two-tailed unpaired t-test) are designated by differentletters. As with bone area, ActRIIB(R64 20-134)-mFc prevented theprogressive decrease in bone mineral content observed under ORXconditions and led to significantly increased bone mineral content undergonad-intact conditions.

FIG. 14 shows whole-body bone mineral density vs. time in mice as afunction of ORX and ActRIIB(R64 20-134)-mFc treatment. Measurements weremade by dual energy X-ray absorptiometry (DEXA) analysis. Data shown aremeans (n=10 per group), and Day 47 means that differ significantly(P<0.05, two-tailed unpaired t-test) are designated by differentletters. ActRIIB(R64 20-134)-mFc increased bone mineral density underORX conditions but not gonad-intact conditions.

FIG. 15 shows bone volume fraction in murine tibia as a function of ORXand ActRIIB(R64 20-134)-mFc treatment for 71 days. Measurements weremade by micro-computed tomography (micro-CT). Data shown are means±SEM(n=7 per group), and those that differ significantly (P<0.05, two-tailedunpaired t-test) are designated by different letters. In ORX mice,ActRIIB(R64 20-134)-mFc increased bone volume fraction markedly comparedto vehicle, restoring this endpoint to levels typical in gonad-intactmice treated with vehicle. ActRIIB(R64 20-134)-mFc increased thisendpoint in gonad-intact mice by a similar magnitude.

FIG. 16 shows trabecular number in murine tibia as a function of ORX andActRIIB(R64 20-134)-mFc treatment for 71 days. Measurements were made bymicro-CT and expressed as the mean number of trabeculae per mm (ofrandomly positioned line segments through the tissue). Data shown aremeans±SEM (n=7 per group), and those that differ significantly (P<0.05,two-tailed unpaired t-test) are designated by different letters. In ORXmice, ActRIIB(R64 20-134)-mFc doubled the trabecular number observedwith vehicle, restoring this endpoint to levels typical in gonad-intactmice treated with vehicle. ActRIIB(R64 20-134)-mFc increased thisendpoint in gonad-intact mice by a similar magnitude.

FIG. 17 shows trabecular thickness in murine tibia as a function of ORXand ActRIIB(R64 20-134)-mFc treatment for 71 days. Measurements weremade by micro-CT. Data shown are means±SEM (n=7 per group), and thosethat differ significantly (P<0.05, two-tailed unpaired t-test) aredesignated by different letters. In ORX mice, ActRIIB(R64 20-134)-mFcincreased trabecular thickness as compared with vehicle, restoring thisendpoint to levels typical in gonad-intact mice treated with vehicle.ActRIIB(R64 20-134)-mFc increased this endpoint in gonad-intact mice bya similar percentage.

FIG. 18 shows trabecular separation in murine tibia as a function of ORXand ActRIIB(R64 20-134)-mFc treatment for 71 days. Measurements weremade by micro-CT. Data shown are means±SEM (n=7 per group), and thosethat differ significantly (P<0.05, two-tailed unpaired t-test) aredesignated by different letters. In ORX mice, ActRIIB(R64 20-134)-mFcdecreased trabecular separation as compared with vehicle, restoring thisendpoint to levels typical in gonad-intact mice treated with vehicle.ActRIIB(R64 20-134)-mFc decreased this endpoint in gonad-intact mice bya similar percentage.

FIG. 19 shows tibial morphology in mice as a function of ORX andActRIIB(R64 20-134)-mFc treatment for 71 days. Images of trabecular bonein the proximal tibia were obtained by micro-CT. Scale bar=100 μm.Tibial morphology in ORX mice treated with ActRIIB(R64 20-134)-mFcclosely resembled that in vehicle-treated gonad-intact mice.

FIG. 20 shows fat tissue mass vs. time in mice as a function of ORX andActRIIB(R64 20-134)-mFc treatment. Measurements were made by NMR. Datashown are means (n=10 per group), and Day 71 means that differsignificantly (P<0.05, two-tailed unpaired t-test) are designated bydifferent letters. Fat mass in vehicle-treated ORX mice tripled over thecourse of the study, and ActRIIB(R64 20-134)-mFc treatment in ORX micecut this increase by more than 60%, restoring this endpoint to levelsobserved in gonad-intact controls. ActRIIB(R64 20-134)-mFc decreasedthis endpoint in gonad-intact mice by a similar percentage.

FIG. 21 shows adipocyte histology in ORX mice treated with vehicle (TBS)or ActRIIB(R64 20-134)-mFc for 71 days. Sections were stained withhematoxylin and eosin. Magnification=10×. ActRIIB-mFc reduced adipocytesize noticeably in subcutaneous and epididymal fat depots but not ininterscapular brown fat.

FIG. 22 shows serum adiponectin concentrations in mice as a function ofORX and ActRIIB(R64 20-134)-mFc treatment for 71 days. ELISAmeasurements detect all main oligomeric isoforms (total adiponectin).Data shown are means±SEM (n=10 per group), and those that differsignificantly (P<0.05, two-tailed unpaired t-test) are designated bydifferent letters. In both ORX and gonad-intact mice, ActRIIB(R6420-134)-mFc increased circulating adiponectin concentrationssignificantly compared to their vehicle-treated counterparts.

FIG. 23 shows serum leptin concentrations in mice as a function of ORXand ActRIIB(R64 20-134)-mFc treatment for 71 days. Data shown aremeans±SEM (n=10 per group), and those that differ significantly (P<0.05,two-tailed unpaired t-test) are designated by different letters. In bothORX and gonad-intact mice, ActRIIB(R64 20-134)-mFc reduced circulatingleptin concentrations significantly compared to their vehicle-treatedcounterparts.

FIG. 24 shows serum levels of adiponectin in mice as a function of dietand ActRIIB-hFc treatment for 60 days. ELISA measurements detect allmain oligomeric isoforms (total adiponectin), and data are means±SEM;n=7-10 per group; **, p<0.01; ***, p<0.001. In mice fed a high-fat diet,ActRIIB(20-134)-hFc increased circulating adiponectin concentrations bymore than 50% to match those in standard-diet controls, whileActRIIB(25-131)-hFc increased circulating adiponectin concentrations bymore than 75% to significantly exceed those in standard-diet controls.

FIG. 25 shows levels of adiponectin mRNA in epididymal white fat of miceas a function of diet and ActRIIB(25-131)-hFc treatment for 60 days.RT-PCR data (in relative units, RU) are means±SEM; n=7 per group; *,p<0.05. In mice fed a high-fat diet, ActRIIB(25-131)-hFc increasedadiponectin mRNA levels by more than 60%, thus contributing to elevatedconcentrations of circulating adiponectin in these mice.

DETAILED DESCRIPTION 1. Overview

In certain aspects, the present invention relates to adiponectin (alsoknown as Acrp30, AdipoQ, apM1, and GBP28), a polypeptide hormone (247amino acids) released from adipocytes in multimeric form. Adiponectinacts through two receptors: AdipoR1, which is expressed in skeletalmuscle, vascular endothelial cells, cardiomyocytes, and pancreatic βcells, and AdipoR2, which is expressed in liver and endothelial cells.Whereas other prominent adipokines (adipocyte-derived hormones) such asleptin and resistin are considered proinflammatory, adiponectin exertsanti-inflammatory effects that seem to serve a counterbalancing role(Szmitko et al., 2007, Am J Physiol Heart Circ Physiol 292:H1655-H1663).Circulating levels of adiponectin vary inversely with adipose mass, andthus low adiponectin levels (hypoadiponectinemia) may partially mediatethe increased risk of cardiovascular disease and type 2 diabetesassociated with obesity. However, hypoadiponectinemia is associated withincreased risk of cardiovascular disease and diabetes even in nonobeseindividuals (Pellme et al., 2003, Diabetes 52:1182-1186; Im et al.,2006, Metabolism 55:1546-1550). Thus the state having abnormally lowadiponectin levels is understood to represent an independentdysfunctional state, and may also identify subset of patients afflictedwith another condition (e.g., type II diabetes, obesity orcardiovascular disease) that are particularly amenable to treatment withan agent described herein.

Evidence suggests a causal protective role for adiponectin in thedevelopment of cardiovascular disease. Adiponectin levels in patientswith coronary heart disease or cerebrovascular disease are lower than inhealthy controls (Hotta et al., 2000, Arterioscler Thromb Vasc Biol20:1595-1599; Kumada et al., 2003, Arterioscler Thromb Vasc Biol23:85-89; Pischon et al., 2004, JAMA 291:1730-1737) and vary inverselywith the severity of disease. Moreover, administration of adiponectininhibits development of atherosclerosis in animal models (Okamoto etal., 2002, Circulation 106:2767-2770), providing evidence for a causalrelationship between adiponectin levels and cardiovascular disease. Asdescribed in the Examples, ActRIIB-Fc fusion proteins can be used toincrease circulating adiponectin levels in diverse mouse models.Therefore, ActRIIB-derived agents and other compounds that inhibitActRIIB signaling can be used to treat or prevent hypoadiponectinemiaand to treat a subset of patients having a condition such ascardiovascular disease, diabetes, and obesity coupled with lowadiponectin.

Low adiponectin, or hypoadiponectinemia, may be understood as the set ofpatients in the lowest quintile of adiponectin levels (below about 10.5mg/L per Pischon et al. JAMA 2004; 291: 1730-1737), and preferably below4.0 mg/L or below 2.5 mg/L (see also Im et al. Metabolism 2006;55:1546-1550; Kumada et al. Arterioscler Thromb Vasc Biol 2003;23:85-89; Ryo et al. Circ J 2004; 68:975-981; Tsukinoki et al. LipidsHealth Dis 2005; 4:27). Values may be slightly higher in women than inmen.

In certain aspects, the present invention relates to ActRIIBpolypeptides. As used herein, the term “ActRIIB” refers to a family ofactivin receptor type IIB (ActRIIB) proteins and ActRIIB-relatedproteins, derived from any species. Members of the ActRIIB family aregenerally all transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine kinasespecificity. The amino acid sequence of human ActRIIB precursor protein,including the native leader, is illustrated in FIG. 2 (SEQ ID NO: 2) andis used throughout this disclosure as the base sequence for numberingthe amino acids of any of the various truncations, mature forms, andvariants of ActRIIB.

The term “ActRIIB polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of an ActRIIB familymember as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Forexample, ActRIIB polypeptides include polypeptides derived from thesequence of any known ActRIIB having a sequence at least about 80%identical to the sequence of an ActRIIB polypeptide, and preferably atleast 85%, 90%, 95%, 97%, 99% or greater identity.

In a specific embodiment, the invention relates to soluble ActRIIBpolypeptides. As described herein, the term “soluble ActRIIBpolypeptide” generally refers to polypeptides comprising anextracellular domain of an ActRIIB protein. The term “soluble ActRIIBpolypeptide,” as used herein, includes any naturally occurringextracellular domain of an ActRIIB protein as well as any variantsthereof (including mutants, fragments and peptidomimetic forms) thatretain a useful activity. For example, the extracellular domain of anActRIIB protein binds to a ligand and is generally soluble. Examples ofsoluble ActRIIB polypeptides include ActRIIB soluble polypeptidesillustrated in FIG. 1 (SEQ ID NO: 1) as well as SEQ ID Nos. 5 and 23.Other examples of soluble ActRIIB polypeptides comprise a signalsequence in addition to the extracellular domain of an ActRIIB protein,see Example 1. The signal sequence can be a native signal sequence of anActRIIB, or a signal sequence from another protein, such as a tissueplasminogen activator (TPA) signal sequence or a honey bee melatin (HBM)signal sequence.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell. Biol. 1:169-178). These type I and type II receptors areall transmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type 1 and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIA and ActRIIB can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell. 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54.

In certain embodiments, the present invention relates to antagonizing aligand of ActRIIB receptors (also referred to as an ActRIIB ligand) witha subject ActRIIB polypeptide (e.g., a soluble ActRIIB polypeptide).Thus, compositions and methods of the present invention are useful fortreating disorders associated with abnormal activity of one or moreligands of ActRIIB receptors. Exemplary ligands of ActRIIB receptorsinclude some TGF-β family members, such as activin, Nodal, GDF8, GDF11,and BMP7.

Activins are dimeric polypeptide growth factors and belong to theTGF-beta superfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). In the TGF-beta superfamily, activins areunique and multifunctional factors that can stimulate hormone productionin ovarian and placental cells, support neuronal cell survival,influence cell-cycle progress positively or negatively depending on celltype, and induce mesodermal differentiation at least in amphibianembryos (DePaolo et al., 1991, Proc SocEp Biol Med. 198:500-512; Dysonet al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). Moreover, erythroid differentiation factor (EDF) isolatedfrom the stimulated human monocytic leukemic cells was found to beidentical to activin A (Murata et al., 1988, PNAS, 85:2434). It wassuggested that activin A acts as a natural regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin, which are describedbelow.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

Bone morphogenetic protein (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide array of physiological processes. Forexample, BMP7 may be the osteoinductive factor responsible for thephenomenon of epithelial osteogenesis. It is also found that BMP7 playsa role in adipocyte differentiation and brown fat formation. Likeactivin, BMP7 binds to type II receptors, ActRIIA and IIB. However, BMP7and activin recruit distinct type I receptors into heteromeric receptorcomplexes. The major BMP7 type I receptor observed was ALK2, whileactivin bound exclusively to ALK4 (ActRIIB). BMP7 and activin eliciteddistinct biological responses and activated different Smad pathways(Macias-Silva et al., 1998, J Biol. Chem. 273:25628-36).

In certain aspects, the present invention relates to the use of certainActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) to antagonizethe signaling of ActRIIB ligands generally, in any process associatedwith ActRIIB activity. Optionally, ActRIIB polypeptides of the inventionmay antagonize one or more ligands of ActRIIB receptors, such asactivins, Nodal, GDF8, GDF11, and BMP7, and may therefore be useful inthe treatment of additional disorders.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRIIB Polypeptides

In certain aspects, the invention relates to ActRIIB variantpolypeptides (e.g., soluble ActRIIB polypeptides). Optionally, thefragments, functional variants, and modified forms have similar or thesame biological activities of their corresponding wild-type ActRIIBpolypeptides. For example, an ActRIIB variant of the invention may bindto and inhibit function of an ActRIIB ligand (e.g., activin A, activinAB, activin B, Nodal, GDF8, GDF11 or BMP7). Optionally, an ActRIIBpolypeptide modulates growth of tissues such as bone, cartilage, muscleor fat. Examples of ActRIIB polypeptides include human ActRIIB precursorpolypeptide (SEQ ID NO: 2), and soluble human ActRIIB polypeptides(e.g., SEQ ID NOs: 1, 2, 5, 12, 23 and 26).

The disclosure identifies functionally active portions and variants ofActRIIB. Applicants have ascertained that an Fc fusion protein havingthe sequence disclosed by Hilden et al. (Blood. 1994 Apr. 15;83(8):2163-70), which has an Alanine at the position corresponding toamino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity foractivin and GDF-11. By contrast, the same Fc fusion protein with anArginine at position 64 (R64) has an affinity for activin and GDF-11 inthe low nanomolar to high picomolar range. Therefore, a sequence with anR64 is used as the wild-type reference sequence for human ActRIIB inthis disclosure.

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that adeletion of the proline knot at the C-terminus of the extracellulardomain of ActRIIB reduced the affinity of the receptor for activin. Datapresented here shows that an ActRIIB-Fc fusion protein containing aminoacids 20-119 of SEQ ID NO:2, “ActRIIB(20-119)-Fc” has reduced binding toGDF-11 and activin relative to an ActRIIB(20-134)-Fc, which includes theproline knot region and the complete juxtamembrane domain. However, anActRIIB(20-129)-Fc protein retains similar but somewhat reduced activityrelative to the wild type, even though the proline knot region isdisrupted. Thus, ActRIIB extracellular domains that stop at amino acid134, 133, 132, 131, 130 and 129 are all expected to be active, butconstructs stopping at 134 or 133 may be most active. Similarly,mutations at any of residues 129-134 are not expected to alter ligandbinding affinity by large margins. In support of this, mutations of P129and P130 do not substantially decrease ligand binding. Therefore, anActRIIB-Fc fusion protein may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 are expectedto have reduced ligand binding. Amino acid 119 is poorly conserved andso is readily altered or truncated. Forms ending at 128 or later retainligand binding activity. Forms ending at or between 119 and 127 willhave an intermediate binding ability. Any of these forms may bedesirable to use, depending on the clinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before will retain ligand binding activity. Amino acid29 represents the initial cysteine. An alanine to asparagine mutation atposition 24 introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding. This confirms that mutations inthe region between the signal cleavage peptide and the cysteinecross-linked region, corresponding to amino acids 20-29 are welltolerated. In particular, constructs beginning at position 20, 21, 22,23 and 24 will retain activity, and constructs beginning at positions25, 26, 27, 28 and 29 are also expected to retain activity.

Taken together, an active portion of ActRIIB comprises amino acids29-109 of SEQ ID NO:2, presented here as SEQ ID NO: 26:

Constructs may, for example, begin at a residue corresponding to aminoacids 20-29 and end at a position corresponding to amino acids 109-134of SEQ ID NO: 2. Other examples include constructs that begin at aposition from 20-29 or 21-29 and end at a position from 119-134, 119-133or 129-134, 129-133. Other examples include constructs that begin at aposition from 20-24 (or 21-24, or 22-25) and end at a position from109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or 129-133).Variants within these ranges are also contemplated, particularly thosehaving at least 80%, 85%, 90%, 95% or 99% identity to the correspondingportion of SEQ ID NO:4.

The disclosure includes the results of an analysis of composite ActRIIBstructures, shown in FIG. 5, demonstrating that the ligand bindingpocket is defined by residues Y31, N33, N35, L38 through T41, E47, E50,Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87,A92, and E94 through F101. At these positions, it is expected thatconservative mutations will be tolerated, although a K74A mutation iswell-tolerated, as are R40A, K55A, F82A and mutations at position L79.R40 is a K in Xenopus, indicating that basic amino acids at thisposition will be tolerated. Q53 is R in bovine ActRIIB and K in XenopusActRIIB, and therefore amino acids including R, K, Q, N and H will betolerated at this position. Thus, a general formula for an activeActRIIB variant protein is one that comprises amino acids 29-109, butoptionally beginning at a position ranging from 20-24 or 22-25 andending at a position ranging from 129-134, and comprising no more than1, 2, 5, 10 or 15 conservative amino acid changes in the ligand bindingpocket, and zero, one or more non-conservative alterations at positions40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket. Such aprotein may retain greater than 80%, 90%, 95% or 99% sequence identityto the sequence of amino acids 29-109 of SEQ ID NO:4. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73. An asparagine to alaninealteration at position 65 (N65A) actually improves ligand binding in theA64 background, and is thus expected to have no detrimental effect onligand binding in the R64 background. This change probably eliminatesglycosylation at N65 in the A64 background, thus demonstrating that asignificant change in this region is likely to be tolerated. While anR64A change is poorly tolerated, R64K is well-tolerated, and thusanother basic residue, such as H may be tolerated at position 64.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also, highly conserverd. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB variant may include one or more amino acids atcorresponding positions from the sequence of another vertebrate ActRIIB,or may include a residue that is similar to that in the human or othervertebrate sequence. The following examples illustrate this approach todefining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB,and so this position may be altered, and optionally may be altered toanother hydrophobic residue, such as V, I or F, or a non-polar residuesuch as A. E52 is a K in Xenopus, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus,indicating that a wide structural variation is tolerated at thisposition, with polar residues favored, such as S, K, R, E, D, H, G, P, Gand Y. F108 is a Yin Xenopus, and therefore Y or other hydrophobicgroup, such as 1, V or L should be tolerated. E111 is K in Xenopus,indicating that charged residues will be tolerated at this position,including D, R, K and H, as well as Q and N. R112 is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 is relatively poorly conserved, and appearsas P in rodents and V in Xenopus, thus essentially any amino acid shouldbe tolerated at this position.

Further N-linked glycosylation sites (N-X-S/T) may be introduced intothe ActRIIb sequence. By introducing an asparagine at position 24 (A24Nconstruct), an NXT sequence is created. Other NX(T/S) sequences arefound at 42-44 (NQS) and 65-67 (NSS), although the latter may not beefficiently glycosylated with the R at position 64. N-X-S/T sequencesmay be generally introduced at positions outside the ligand bindingpocket defined in FIG. 12. Particularly suitable sites for theintroduction of non-endogenous N-X-S/T sequences include amino acids20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. N-X-S/T sequences mayalso be introduced into the linker between the ActRIIB sequence and theFc or other fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Thus, desirable alterations thatwould create an N-linked glycosylation site are: A24N, R64N, S67N(possibly combined with an N65A alteration), E106N, R112N, G120N, E123N,P129N, A132N, R112S and R112T. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S. Thus thealterations S67T and S44T are contemplated. Likewise, in an A24Nvariant, an S26T alteration may be used. Accordingly, an ActRIIB variantmay include one or more additional, non-endogenous N-linkedglycosylation consensus sequences.

The variations described may be combined in various ways. Additionally,the results of mutagenesis program described previously in WO2006/012627 and WO 2008/097541 indicate that there are amino acidpositions in ActRIIb that are often beneficial to conserve. Theseinclude position 64 (basic amino acid), position 80 (acidic orhydrophobic amino acid), position 78 (hydrophobic, and particularlytryptophan), position 37 (acidic, and particularly aspartic or glutamicacid), position 56 (basic amino acid), position 60 (hydrophobic aminoacid, particularly phenylalanine or tyrosine). Thus the disclosureprovides a framework of amino acids that may be conserved. Otherpositions that may be desirable to conserve are as follows: position 52(acidic amino acid), position 55 (basic amino acid), position 81(acidic), 98 (polar or charged, particularly E, D, R or K).

In certain embodiments, isolated fragments of the ActRIIB polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding an ActRIIBpolypeptide (e.g., SEQ ID NOs: 3 and 4). In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function, forexample, as antagonists (inhibitors) or agonists (activators) of anActRIIB protein or an ActRIIB ligand.

In certain embodiments, a functional variant of the ActRIIB polypeptideshas an amino acid sequence that is at least 75% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 5, 12, 23 and 26. Incertain cases, the functional variant has an amino acid sequence atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 5, 12, 23 and 26.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of an ActRIIB polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).Modified ActRIIB polypeptides can also be produced, for instance, byamino acid substitution, deletion, or addition. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of an ActRIIB polypeptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant ActRIIB polypeptide to produce a response in cells in afashion similar to the wild-type ActRIIB polypeptide, or to bind to oneor more ligands, such as activin, GDF-11 or myostatin in a fashionsimilar to wild type.

In certain specific embodiments, the present invention contemplatesmaking mutations in the extracellular domain (also referred to asligand-binding domain) of an ActRIIB polypeptide such that the variant(or mutant) ActRIIB polypeptide has altered ligand-binding activities(e.g., binding affinity or binding specificity). In certain cases, suchvariant ActRIIB polypeptides have altered (elevated or reduced) bindingaffinity for a specific ligand. In other cases, the variant ActRIIBpolypeptides have altered binding specificity for their ligands.

For example, the disclosure provides variant ActRIIB polypeptides thatpreferentially bind to GDF8/GDF11 relative to activins. The disclosurefurther establishes the desirability of such polypeptides for reducingoff-target effects, although such selective variants may be lessdesirable for the treatment of severe diseases where very large gains inmuscle mass may be needed for therapeutic effect and where some level ofoff-target effect is acceptable. For example, amino acid residues of theActRIIB protein, such as E39, K55, Y60, K74, W78, D80, and F101, are inthe ligand-binding pocket and mediate binding to its ligands such asactivin and GDF8. Thus, the present invention provides an alteredligand-binding domain (e.g., GDF8-binding domain) of an ActRIIBreceptor, which comprises one or more mutations at those amino acidresidues. Optionally, the altered ligand-binding domain can haveincreased selectivity for a ligand such as GDF8 relative to a wild-typeligand-binding domain of an ActRIIB receptor. To illustrate, thesemutations increase the selectivity of the altered ligand-binding domainfor GDF8 over activin. Optionally, the altered ligand-binding domain hasa ratio of K_(d) for activin binding to K_(d) for GDF8 binding that isat least 2, 5, 10, or even 100 fold greater relative to the ratio forthe wild-type ligand-binding domain. Optionally, the alteredligand-binding domain has a ratio of IC₅₀ for inhibiting activin to IC₅₀for inhibiting GDF8 that is at least 2, 5, 10, or even 100 fold greaterrelative to the wild-type ligand-binding domain. Optionally, the alteredligand-binding domain inhibits GDF8 with an IC₅₀ at least 2, 5, 10, oreven 100 times less than the IC₅₀ for inhibiting activin.

As a specific example, the positively-charged amino acid residue Asp(D80) of the ligand-binding domain of ActRIIB can be mutated to adifferent amino acid residue such that the variant ActRIIB polypeptidepreferentially binds to GDF8, but not activin. Preferably, the D80residue is changed to an amino acid residue selected from the groupconsisting of: a uncharged amino acid residue, a negative amino acidresidue, and a hydrophobic amino acid residue. As a further specificexample, the hydrophobic residue, L79, can be altered to the acidicamino acids aspartic acid or glutamic acid to greatly reduce activinbinding while retaining GDF11 binding. As will be recognized by one ofskill in the art, most of the described mutations, variants ormodifications may be made at the nucleic acid level or, in some cases,by post translational modification or chemical synthesis. Suchtechniques are well known in the art.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIB polypeptides so as to alter the glycosylationof the polypeptide. Exemplary glycosylation sites in ActRIIBpolypeptides are illustrated in FIG. 2. Such mutations may be selectedso as to introduce or eliminate one or more glycosylation sites, such asO-linked or N-linked glycosylation sites. Asparagine-linkedglycosylation recognition sites generally comprise a tripeptidesequence, asparagine-X-threonine (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ActRIIB polypeptide (for O-linked glycosylation sites). Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Another means ofincreasing the number of carbohydrate moieties on an ActRIIB polypeptideis by chemical or enzymatic coupling of glycosides to the ActRIIBpolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups such as those of cysteine; (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated byreference herein. Removal of one or more carbohydrate moieties presenton an ActRIIB polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of the ActRIIB polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRIIB polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIB polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIB proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of an ActRIIB polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying functional variantsequences. The purpose of screening such combinatorial libraries may beto generate, for example, ActRIIB polypeptide variants which havealtered properties, such as altered pharmacokinetics, or altered ligandbinding. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIBpolypeptide variant may be screened for ability to bind to an ActRIIBpolypeptide, to prevent binding of an ActRIIB ligand to an ActRIIBpolypeptide.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring ActRIIB polypeptide. Suchvariant proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols. Likewise, mutagenesis can give rise tovariants which have intracellular half-lives dramatically different thanthe corresponding a wild-type ActRIIB polypeptide. For example, thealtered protein can be rendered either more stable or less stable toproteolytic degradation or other processes which result in destructionof, or otherwise inactivation of a native ActRIIB polypeptide. Suchvariants, and the genes which encode them, can be utilized to alterActRIIB polypeptide levels by modulating the half-life of the ActRIIBpolypeptides. For instance, a short half-life can give rise to moretransient biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant ActRIIB polypeptidelevels within the cell.

In certain embodiments, the ActRIIB polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIB polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIB polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of an ActRIIB polypeptide may be tested asdescribed herein for other ActRIIB polypeptide variants. When an ActRIIBpolypeptide is produced in cells by cleaving a nascent form of theActRIIB polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIB polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIBpolypeptides include fusion proteins having at least a portion of theActRIIB polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (e.g., an Fc),maltose binding protein (MBP), or human serum albumin. A fusion domainmay be selected so as to confer a desired property. For example, somefusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ActRIIBpolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ActRIIBpolypeptide is fused with a domain that stabilizes the ActRIIBpolypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction, such as further stimulation of muscle growth).

The following is a specific example of Fc domains that may be used(e.g., SEQ ID NO: 13).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDCPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

The Fc domain may have one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIB polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIB polypeptide. The ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In the case of fusion proteins, an ActRIIB polypeptide may be fused to astabilizer domain such as an IgG molecule (e.g., an Fc domain). As usedherein, the term “stabilizer domain” not only refers to a fusion domain(e.g., Fc) as in the case of fusion proteins, but also includesnonproteinaceous modifications such as a polyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIB polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, ActRIIB polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such ActRIIB polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the ActRIIB polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified ActRIIB polypeptidesmay be produced by digestion of naturally occurring or recombinantlyproduced full-length ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. Alternatively, such ActRIIB polypeptides may be produced fromnaturally occurring or recombinantly produced full-length ActRIIBpolypeptides such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRIIB Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIB polypeptides (e.g., solubleActRIIB polypeptides), including any of the variants disclosed herein.For example, SEQ ID NO: 4 encodes a naturally occurring ActRIIBprecursor polypeptide, while SEQ ID NO: 3 encodes a soluble ActRIIBpolypeptide. The subject nucleic acids may be single-stranded or doublestranded. Such nucleic acids may be DNA or RNA molecules. These nucleicacids are may be used, for example, in methods for making ActRIIBpolypeptides or as direct therapeutic agents (e.g., in a gene therapyapproach).

In certain aspects, the subject nucleic acids encoding ActRIIBpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 3. Variant nucleotide sequences include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will, therefore, include codingsequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NO: 4.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 3, 10 and 24. One of ordinary skillin the art will appreciate that nucleic acid sequences complementary toSEQ ID NO: 3, and variants of SEQ ID NO: 3 are also within the scope ofthis invention. In further embodiments, the nucleic acid sequences ofthe invention can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 3, 10 or 24, complementsequence of SEQ ID NO: 3, or fragments thereof. As discussed above, oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Forexample, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 3 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIB polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRIIB polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIB polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIB polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCl-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIB polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 3, 4, 10or 24) for one or more of the subject ActRIIB polypeptide. The host cellmay be any prokaryotic or eukaryotic cell. For example, an ActRIIBpolypeptide of the invention may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIB polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIB polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIB polypeptide to occur. The ActRIIB polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIBpolypeptide. Alternatively, the ActRIIB polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIB polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the ActRIIB polypeptides. In a preferred embodiment, theActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIBpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIB polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Antibodies

Another aspect of the invention pertains to antibodies. An antibody thatis specifically reactive with an ActRIIB polypeptide (e.g., a solubleActRIIB polypeptide) and which binds competitively with the ActRIIBpolypeptide may be used as an antagonist of ActRIIB polypeptideactivities. For example, by using immunogens derived from an ActRIIBpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory. Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRIIB polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRIIB polypeptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIB polypeptide, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIBpolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject ActRIIBpolypeptide. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for an ActRIIB polypeptide conferred by at least one CDRregion of the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain preferred embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments, the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to anActRIIB polypeptide may comprise administering to a mouse an amount ofan immunogenic composition comprising the ActRIIB polypeptide effectiveto stimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monocolonal antibody that binds specificallyto the ActRIIB polypeptide. Once obtained, a hybridoma can be propagatedin a cell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the ActRIIB polypeptide. The monoclonal antibody may bepurified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIB polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure provides antibodies that bind to asoluble ActRIIB polypeptide. Such antibodies may be generated much asdescribed above, using a soluble ActRIIB polypeptide or fragment thereofas an antigen. Antibodies of this type can be used, e.g., to detectActRIIB polypeptides in biological samples and/or to monitor solubleActRIIB polypeptide levels in an individual. In certain cases, anantibody that specifically binds to a soluble ActRIIB polypeptide can beused to modulate activity of an ActRIIB polypeptide and/or an ActRIIBligand, thereby regulating (promoting or inhibiting) growth of tissues,such as bone, cartilage, muscle, fat, and neurons.

5. Screening Assays

In certain aspects, the present invention relates to the use of thesubject ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) toidentify compounds (agents) which are agonist or antagonists of theActRIIB polypeptides. Compounds identified through this screening can betested in tissues such as bone, cartilage, muscle, fat, and/or neurons,to assess their ability to modulate tissue growth in vitro. Optionally,these compounds can further be tested in animal models to assess theirability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting the ActRIIB polypeptides. Incertain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb ActRIIB-mediated effects ongrowth of bone, cartilage, muscle, fat, and/or neurons. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIB polypeptide toits binding partner, such as an ActRIIB ligand (e.g., activin, Nodal,GDF8, GDF11 or BMP7). Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRIIB polypeptide to its bindingprotein such as an ActRIIB ligand. In a further embodiment, thecompounds can be identified by their ability to interact with an ActRIIBpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIBpolypeptide and its binding protein (e.g., an ActRIIB ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIB polypeptide which is ordinarily capable of binding toan ActRIIB ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRIIB polypeptide is then added acomposition containing an ActRIIB ligand. Detection and quantificationof ActRIIB/ActRIIB ligand complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the ActRIIB polypeptide and its binding protein. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, isolated and purifiedActRIIB ligand is added to a composition containing the ActRIIBpolypeptide, and the formation of ActRIIB/ActRIIB ligand complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the ActRIIB polypeptide and its bindingprotein may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labeled proteins such as radiolabeled (e.g., ³²P,³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), or enzymaticallylabeled ActRIIB polypeptide or its binding protein, by immunoassay, orby chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRIIB polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRIIB polypeptideand its binding protein. See for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIB polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRIIB polypeptide of the invention. Theinteraction between the compound and the ActRIIB polypeptide may becovalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an ActRIIBpolypeptide. This may include a solid phase or fluid phase bindingevent. Alternatively, the gene encoding an ActRIIB polypeptide can betransfected with a reporter system (e.g., β-galactosidase, luciferase,or green fluorescent protein) into a cell and screened against thelibrary preferably by a high throughput screening or with individualmembers of the library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor stimulating muscle growth and increasing muscle mass, for example,by antagonizing functions of an ActRIIB polypeptide and/or an ActRIIBligand. Therefore, any compound identified can be tested in whole cellsor tissues, in vitro or in vivo, to confirm their ability to modulatemuscle growth. Various methods known in the art can be utilized for thispurpose. For example, methods of the invention are performed such thatthe signal transduction through an ActRIIB protein activated by bindingto an ActRIIB ligand (e.g., GDF8) has been reduced or inhibited. It willbe recognized that the growth of muscle tissue in the organism wouldresult in an increased muscle mass in the organism as compared to themuscle mass of a corresponding organism (or population of organisms) inwhich the signal transduction through an ActRIIB protein had not been soeffected.

For example, the effect of the ActRIIB polypeptides or test compounds onmuscle cell growth/proliferation can be determined by measuring geneexpression of Pax-3 and Myf-5 which are associated with proliferation ofmyogenic cells, and gene expression of MyoD which is associated withmuscle differentiation (e.g., Amthor et al., Dev Biol. 2002,251:241-57). It is known that GDF8 down-regulates gene expression ofPax-3 and Myf-5, and prevents gene expression of MyoD. The ActRIIBpolypeptides or test compounds are expected to antagonize this activityof GDF8. Another example of cell-based assays includes measuring theproliferation of myoblasts such as C(2)C(12) myoblasts in the presenceof the ActRIIB polypeptides or test compounds (e.g., Thomas et al., JBiol. Chem. 2000, 275:40235-43).

The present invention also contemplates in vivo assays to measure musclemass and strength. For example, Whittemore et al. (Biochem Biophys ResCommun. 2003, 300:965-71) discloses a method of measuring increasedskeletal muscle mass and increased grip strength in mice. Optionally,this method can be used to determine therapeutic effects of testcompounds (e.g., ActRIIB polypeptides) on muscle diseases or conditions,for example those diseases for which muscle mass is limiting.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) bone formation and increasingbone mass. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate bone or cartilage growth. Various methods known in the art canbe utilized for this purpose.

For example, the effect of the ActRIIB polypeptides or test compounds onbone or cartilage growth can be determined by measuring induction ofMsx2 or differentiation of osteoprogenitor cells into osteoblasts incell based assays (see, e.g., Daluiski et al., Nat. Genet. 2001,27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Another exampleof cell-based assays includes analyzing the osteogenic activity of thesubject ActRIIB polypeptides and test compounds in mesenchymalprogenitor and osteoblastic cells. To illustrate, recombinantadenoviruses expressing an ActRIIB polypeptide were constructed toinfect pluripotent mesenchymal progenitor C3H10T1/2 cells,preosteoblastic C2C12 cells, and osteoblastic TE-85 cells. Osteogenicactivity is then determined by measuring the induction of alkalinephosphatase, osteocalcin, and matrix mineralization (see, e.g., Cheng etal., J bone Joint Surg Am. 2003, 85-A(8):1544-52).

The present invention also contemplates in vivo assays to measure boneor cartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86(2001) discloses a rat osteoporotic model in which bone repair duringthe early period after fracture is studied. Kubo et al., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a ratosteoporotic model in which bone repair during the late period afterfracture is studied. These references are incorporated by referenceherein in their entirety for their disclosure of rat model for study onosteoporotic bone fracture. In certain aspects, the present inventionmakes use of fracture healing assays that are known in the art. Theseassays include fracture technique, histological analysis, andbiomechanical analysis, which are described in, for example, U.S. Pat.No. 6,521,750, which is incorporated by reference in its entirety forits disclosure of experimental protocols for causing as well asmeasuring the extent of fractures, and the repair process.

In certain aspects, the present invention provides methods and agentsfor controlling weight gain and obesity. At the cellular level,adipocyte proliferation and differentiation is critical in thedevelopment of obesity, which leads to the generation of additional fatcells (adipocytes). Therefore, any compound identified can be tested inwhole cells or tissues, in vitro or in vivo, to confirm their ability tomodulate adipogenesis by measuring adipocyte proliferation ordifferentiation. Various methods known in the art can be utilized forthis purpose. For example, the effect of an ActRIIB polypeptide (e.g., asoluble ActRIIB polypeptide) or test compounds on adipogenesis can bedetermined by measuring differentiation of 3T3-L1 preadipocytes tomature adipocytes in cell based assays, such as, by observing theaccumulation of triacylglycerol in Oil Red O staining vesicles and bythe appearance of certain adipocyte markers such as FABP (aP2/422) andPPARγ2. See, for example, Reusch et al., 2000, Mol Cell Biol.20:1008-20; Deng et al., 2000, Endocrinology. 141:2370-6; Bell et al.,2000, Obes Res. 8:249-54. Another example of cell-based assays includesanalyzing the role of ActRIIB polypeptides and test compounds inproliferation of adipocytes or adipocyte precursor cells (e.g., 3T3-L1cells), such as, by monitoring bromodeoxyuridine (BrdU)-positive cells.See, for example, Pico et al., 1998, Mol Cell Biochem. 189:1-7; Masunoet al., 2003, Toxicol Sci. 75:314-20.

It is understood that the screening assays of the present inventionapply to not only the subject ActRIIB polypeptides and variants of theActRIIB polypeptides, but also any test compounds including agonists andantagonist of the ActRIIB polypeptides. Further, these screening assaysare useful for drug target verification and quality control purposes.

6. Exemplary Therapeutic Uses

In certain embodiments, compositions (e.g., ActRIIB polypeptides) of thepresent invention can be used for treating or preventinghypoadiponectinemia and interrelated conditions. In certain embodiments,the present invention provides methods of treating or preventing anindividual in need thereof through administering to the individual atherapeutically effective amount of an ActRIIB polypeptide as describedabove. These methods are particularly aimed at therapeutic andprophylactic treatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

As demonstrated herein, ActRIIB-Fc administration in vivo promotesexpression of adiponectin in white adipose tissue and increasescirculating adiponectin levels in diverse mouse models. Accordingly,compositions disclosed herein may be used to treat or preventhypoadiponectinemia and associated disorders, including to subset ofpatients with atherosclerosis, ischemic stroke, impaired glucosetolerance, insulin resistance, diabetes type 2, hyperlipidemia,hypertriglyceridemia, or obesity that also exhibit low circulatingadiponectin.

In other related embodiments, soluble ActRIIB polypeptides and othercompositions of the invention can be used as part of treatment orprevention of atherosclerosis, a chronic inflammatory condition in whichartery walls thicken due to the accumulation of fatty deposits, oftenreferred to as plaques. Risk factors for atherosclerosis include aging,diabetes mellitus, dyslipoproteinemia, obesity (abdominal or visceraladiposity), and a sedentary lifestyle.

Soluble ActRIIB polypeptides can also be used for treatment orprevention of lipodystrophic disorders, which tend to be associated withmetabolic syndrome. Severe insulin resistance can result from bothgenetic and acquired forms of lipodystrophy, including in the lattercase human immunodeficiency virus (HIV)-related lipodystrophy inpatients treated with antiretroviral therapy.

In related embodiments, soluble ActRIIB polypeptides and othercompositions of the invention can be used as part of treatment orprevention of diabetes mellitus type II (also known asnon-insulin-dependent diabetes mellitus or adult-onset diabetes), whichis characterized by elevated blood glucose in the context of insulinresistance and relative insulin deficiency. Complex and multifactorialmetabolic changes in diabetes often lead to damage and functionalimpairment of many organs, most importantly the cardiovascular system.Diabetes mellitus type II is often associated with obesity (abdominal orvisceral adiposity), hypertension, elevated cholesterol, and metabolicsyndrome. Important risk factors for diabetes mellitus type II includeaging, high-fat diets, and a sedentary lifestyle.

The subject ActRIIB polypeptides may further be used as a therapeuticagent for slowing or preventing the development of obesity. Thisapproach is confirmed and supported by the data shown herein, whereby anActRIIB-Fc protein was shown to improve metabolic status in mice on ahigh-fat diet.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRIIB polypeptide.

In one specific embodiment, the present invention relates to methods andcompounds for reducing body weight and/or reducing weight gain in ananimal, and more particularly, for treating or ameliorating obesity inpatients at risk for or suffering from obesity. In another specificembodiment, the present invention is directed to methods and compoundsfor treating an animal that is unable to gain or retain weight (e.g., ananimal with a wasting syndrome). Such methods are effective to increasebody weight and/or mass, or to reduce weight and/or mass loss, or toimprove conditions associated with or caused by undesirably low (e.g.,unhealthy) body weight and/or mass.

As demonstrated in WO 2006/012627 and WO 2008/097541, compoundsdisclosed herein stimulate muscle growth. Accordingly, these compoundsmay be particularly useful in diseases or conditions with overlappingmuscle and metabolic dysfunction.

In certain embodiments, compositions (e.g., soluble ActRIIBpolypeptides) of the invention are used as part of a treatment for amuscular dystrophy. The term “muscular dystrophy” refers to a group ofdegenerative muscle diseases characterized by gradual weakening anddeterioration of skeletal muscles and sometimes the heart andrespiratory muscles. Muscular dystrophies are genetic disorderscharacterized by progressive muscle wasting and weakness that begin withmicroscopic changes in the muscle. As muscles degenerate over time, theperson's muscle strength declines. Moreover, declining muscle mass anddiminishing physical activity contribute to an imbalance between caloricintake and energy expenditure, leading to unhealthy storage of excessenergy as white adipose tissue. Exemplary muscular dystrophies that canbe treated with a regimen including the subject ActRIIB polypeptidesinclude: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy(BMD), Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle MuscularDystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD)(also known as Landouzy-Dejerine), Myotonic Dystrophy (MMD) (also knownas Steinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD),Distal Muscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, recent researches demonstrate that blocking or eliminatingfunction of GDF8 (an ActRIIB ligand) in vivo can effectively treat atleast certain symptoms in DMD and BMD patients. Thus, the subjectActRIIB polypeptides may act as GDF8 inhibitors (antagonists), andconstitute an alternative means of blocking the functions of GDF8 and/orActRIIB in vivo in DMD and BMD patients.

ActRIIB polypeptide-induced increased muscle mass might also benefitthose suffering from muscle wasting diseases. Gonzalez-Cadavid et al.(1998, PNAS 95:14938-43) reported that GDF8 expression correlatesinversely with fat-free mass in humans and that increased expression ofthe GDF8 gene is associated with weight loss in men with AIDS wastingsyndrome. By inhibiting the function of GDF8 in AIDS patients, at leastcertain symptoms of AIDS may be alleviated, if not completelyeliminated, thus significantly improving quality of life in AIDSpatients.

Sarcopenia, the loss of muscle with aging is also often associated withmetabolic syndrome, diabetes, arteriosclerosis, dyslipidemia, and otherage-related metabolic conditions. ActRIIB polypeptide-induced musclemass might also benefit those suffering from sarcopenia.

In particular, the present disclosure demonstrates that in certainconditions, such as androgen deprivation, agents disclosed herein can beused to promote muscle and bone formation while decreasing adiposity,and therefore, the disclosure provides methods for treating patientsexhibiting low bone and muscle content and elevated adiposity may beadvantageously treated with soluble ActRIIB polypeptides and otheragents disclosed herein. This may be particularly beneficial in patientsreceiving androgen or estrogen antagonist therapy, elderly patients(e.g., combined sarcopenia, osteoporosis and obesity) and patients witha muscle wasting condition that are also receiving corticosteroidtherapy.

In other embodiments, the present invention provides methods of inducingbone and/or cartilage formation, preventing bone loss, increasing bonemineralization or preventing the demineralization of bone. For example,the subject ActRIIB polypeptides and compounds identified in the presentinvention have application in treating osteoporosis and the healing ofbone fractures and cartilage defects in humans and other animals.ActRIIB polypeptides may be useful in patients that are diagnosed withsubclinical low bone density, as a protective measure against thedevelopment of osteoporosis.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRIIB polypeptide.

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., ActRIIB polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, an ActRIIB polypeptide can be administered aloneor as a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neurons), for example, a sitehaving a tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than theActRIIB polypeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., ActRIIB polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., ActRIIB polypeptides) to a target tissue site, providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the ActRIIB polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., an ActRIIB polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more ActRIIB polypeptidesin combination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIB polypeptides).The various factors will depend upon the disease to be treated. In thecase of muscle disorders, factors may include, but are not limited to,amount of muscle mass desired to be formed, the muscles most affected bydisease, the condition of the deteriorated muscle, the patient's age,sex, and diet, time of administration, and other clinical factors. Theaddition of other known growth factors to the final composition, mayalso affect the dosage. Progress can be monitored by periodic assessmentof muscle growth and/or repair, for example, by strength testing, MRIassessment of muscle size and analysis of muscle biopsies.

In certain embodiments of the invention, one or more ActRIIBpolypeptides can be administered, together (simultaneously) or atdifferent times (sequentially or overlapping). In addition, ActRIIBpolypeptides can be administered with another type of therapeuticagents, for example, a cartilage-inducing agent, a bone-inducing agent,a muscle-inducing agent, a fat-reducing, or a neuron-inducing agent. Thetwo types of compounds may be administered simultaneously or atdifferent times. It is expected that the ActRIIB polypeptides of theinvention may act in concert with or perhaps synergistically withanother therapeutic agent.

In a specific example, a variety of osteogenic, cartilage-inducing andbone-inducing factors have been described, particularly bisphosphonates.See e.g., European Patent Application Nos. 148,155 and 169,016. Forexample, other factors that can be combined with the subject ActRIIBpolypeptides include various growth factors such as epidermal growthfactor (EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIB polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIBpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRIIB polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRIIB polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIB polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIB polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Generation of an ActRIIb-Fc Fusion Protein

Applicants constructed a soluble ActRIIb fusion protein that has theextracellular domain of human ActRIIb fused to a human or mouse Fcdomain with a minimal linker (three glycine amino acids) in between. Theconstructs are referred to as ActRIIb-hFc and ActRIIb-mFc, respectively.

ActRIIb-hFc is shown below as purified from CHO cell lines (SEQ ID NO:5)

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIb-hFc and ActRIIb-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): MKFLVNVALVEMVVYISYIYA (SEQ ID NO: 7) (ii)Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:8) (iii) Native: MGAAAKLAFAVFLISCSSGA. (SEQ ID NO: 9)

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGOPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:10):

A TGGATGCAAT GAAGAGAGCG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTTCGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTCGAGCG CTGCGAAGGC GAGCAGGACAAGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGGAGAACCCCCA GGTCTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCGGTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCACATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGTACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCAAAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGGAGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGGGGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGAGCCTCTCCCT GTCTCCGGGT AAATGA

N-terminal sequencing of the CHO-cell produced material revealed a majorsequence of—GRGEAE (SEQ ID NO: 11). Notably, other constructs reportedin the literature begin with an—SGR . . . sequence.

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange.

ActRIIb-Fc fusion proteins were also expressed in HEK293 cells and COScells. Although material from all cell lines and reasonable cultureconditions provided protein with muscle-building activity in vivo,variability in potency was observed perhaps relating to cell lineselection and/or culture conditions.

Example 2 Generation of ActRIIb-Fc Mutants

Applicants generated a series of mutations in the extracellular domainof ActRIIB and produced these mutant proteins as soluble fusion proteinsbetween extracellular ActRIIB and an Fc domain. The backgroundActRIIB-Fc fusion has the sequence (Fc portion underlined) (SEQ IDNO:12):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Various mutations, including N- and C-terminal truncations, wereintroduced into the background ActRIIB-Fc protein. Based on the datapresented in Example 1, it is expected that these constructs, ifexpressed with a TPA leader, will lack the N-terminal serine. Mutationswere generated in ActRIIB extracellular domain by PCR mutagenesis. AfterPCR, fragments were purified through a Qiagen column, digested with SfoIand AgeI and gel purified. These fragments were ligated into expressionvector pAID4 (see WO2006/012627) such that upon ligation it createdfusion chimera with human IgG 1. Upon transformation into E. coli DH5alpha, colonies were picked and DNAs were isolated. For murineconstructs (mFc), a murine IgG2a was substituted for the human IgG1. Allmutants were sequence verified.

All of the mutants were produced in HEK293T cells by transienttransfection. In summary, in a 500 ml spinner, HEK293T cells were set upat 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated.

Mutants were purified using a variety of techniques, including, forexample, protein A column and eluted with low pH (3.0) glycine buffer.After neutralization, these were dialyzed against PBS.

Mutants were also produced in CHO cells by similar methodology.

Mutants were tested in binding assays and/or bioassays. In someinstances, assays were performed with conditioned medium rather thanpurified proteins.

Example 3 Generation of Truncated Variant ActRIIB(25-131)-hFc

Applicants generated a truncated fusion protein, ActRIIB(25-131)-hFc(FIGS. 7-8), which exhibits effects on muscle that are similar to thoseobserved with ActRIIB(20-134)-hFc (while exhibiting superior effects onother tissues and parameters). ActRIIB(25-131)-hFc was generated usingthe same leader and methodology as described above with respect toActRIIB(20-134)-hFc. The mature ActRIIB(25-131)-hFc protein purifiedafter expression in CHO cells has the sequence shown below (SEQ ID NO:23). Amino acids 1-107 (underlined) are derived from ActRIIB.

(SEQ ID NO: 23) ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWRNSSGTIELVK KGCWLDDFNC YDRQECVATE ENPQVYFCCCEGNFCNERFT HLPEAGGPEV TYEPPPTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEALHNHYTQKSLS LSPGK

The expressed molecule was purified using a series of columnchromatography steps, including for example, three or more of thefollowing, in any order: Protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 4 High-Affinity Ligand Binding by ActRIIB(25-131)-hFc

Affinities of several ligands for ActRIIB(25-131)-hFc and itsfull-length counterpart ActRIIB(20-134)-hFc were evaluated in vitro witha Biacore™ instrument, and the results are summarized in the tablebelow. Kd values were obtained by steady-state affinity fit due to veryrapid association and dissociation of the complex, which preventedaccurate determination of k_(on) and k_(off). ActRIIB(25-131)-hFc boundactivin A, activin B, and GDF11 with high affinity. Intriguingly,ActRIIB(25-131)-hFc appears to show a higher affinity for GDF3 thanActRIIB(20-134)-hFc (data not shown).

Ligand Affinities of ActRIIb-hFc Forms:

Activin A Activin B GDF11 Fusion Construct (e-11) (e-11) (e-11)ActRIIB(20-134)-hFc 1.6 1.2 3.6 ActRIIB(25-131)-hFc 1.8 1.2 3.1

Example 5 Bioassay for GDF-11 and Activin-Mediated Signaling

An A-204 Reporter Gene Assay was used to evaluate the effects ofActRIIB-Fc proteins on signaling by GDF-11 and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100.) SeeFIG. 5. The CAGA12 motif is present in TGF-Beta responsive genes (PAI-1gene), so this vector is of general use for factors signaling throughSmad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 orpGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. In the absence of anyinhibitors, Activin A showed 10 fold stimulation of reporter geneexpression and an ED50˜2 ng/ml. GDF-11: 16 fold stimulation, ED50:˜1.5ng/ml.

ActRIIB(R64, 20-134) is a potent inhibitor of activin, GDF-8 and GDF-11activity in this assay. Variants were tested in this assay as well.

Example 6 GDF-11 Inhibition by N-Terminal and C-Terminal Truncations

Truncations at the N-terminus and C-terminus of the ActRIIB portionActRIIB-Fc (R64, 20-134) were generated and tested for activity asinhibitors of GDF-11 and activin. The activities are shown below (asmeasured in conditioned media):

C-Terminal ActRIIb-hFc Truncations:

IC50 (ng/mL) GDF-11 Activin ActRIIb-hFc (R64, 20-134) 45 22 ActRIIb-hFc(R64, 20-132) 87 32 ActRIIb-hFc (R64, 20-131) 120 44 ActRIIb-hFc (R64,20-128) 130 158

As can be seen, truncations of three (ending with . . . PPT), six(ending with . . . YEP) or more amino acids at the C-terminus causes athreefold or greater decrease in the activity of the molecule. Thetruncation of the final 15 amino acids of the ActRIIB portion causes agreater loss of activity (see WO2006/012627).

Amino terminal truncations were made in the background of an ActRIIb-hFc(R64 20-131) protein. The activities are shown below (as measured inconditioned media):

N-terminal ActRIIb-hFc Truncations:

IC50 (ng/mL) GDF-11 Activin ActRIIb-hFc (R64, 20-131) 183 201 (GRG . ..) ActRIIb-hFc (R64, 21-131) 121 325 (RGE . . .) ActRIIb-hFc (R64,22-131) 71 100 (GEA . . .) ActRIIb-hFc (R64, 23-131) 60 43 (EAE . . .)ActRIIb-hFc (R64, 24-131) 69 105 (AET . . .)

Accordingly, truncations of two, three or four amino acids from theN-terminus lead to the production of a more active protein than theversions with a full-length extracellular domain. Additional experimentsshow that a truncation of five amino acids, ActRIIb-hFc (R64, 25-131)has activity equivalent to the untruncated form, and additionaldeletions at the N-terminus continue to degrade the activity of theprotein. Therefore, optimal constructs will have a C-terminus endingbetween amino acid 133-134 of SEQ ID NO:4 and an N-terminus beginning atamino acids 22-24 of SEQ ID NO:4. An N-terminus corresponding to aminoacids 21 or 25 will give activity that is similar to the ActRIIb-hFc(R64, 20-134) construct, although the protein designated as SEQ ID NO:23 has been characterized as having superior effects in some regards.

Example 7 ActRIIb-Fc Variants, Cell-Based Activity

Activity of ActRIIB-Fc proteins was tested in a cell based assay, asdescribed above. Results are summarized in Table 1, below. Some variantswere tested in different C-terminal truncation constructs. As discussedabove, truncations of five or fifteen amino acids caused reduction inactivity. Remarkably, the L79D and L79E variants showed substantial lossof activin binding while retaining almost wild-type inhibition ofGDF-11.

Soluble ActRIIb-Fc Binding to GDF11 and Activin A:

Portion of ActRIIB GDF11 Activin ActRIIB-Fc (corresponds to aminoInhibition Inhibition Variations acids of SEQ ID NO: 4) ActivityActivity 64R 20-134 +++ +++ (approx. (approx. 10⁻⁸ M K₁) 10⁻⁸ M K₁) 64A20-134 + + (approx. (approx. 10⁻⁶ M K₁) 10⁻⁶ M K₁) 64R 20-129 +++ +++64R K74A 20-134 ++++ ++++ 64R A24N 20-134 +++ +++ 64R A24N 20-119 ++ ++64R A24N 20-119 + + K74A R64 L79P 20-134 + + R64 L79P 20-134 + + K74AR64 L79D 20-134 +++ + R64 L79E 20-134 +++ + R64K 20-134 +++ +++ R64K20-129 +++ +++ R64 P129S 20-134 +++ +++ P130A R64N 20-134 + + + Pooractivity (roughly 1 × 10⁻⁶ K₁) ++ Moderate activity (roughly 1 × 10⁻⁷K₁) +++ Good (wild-type) activity (roughly 1 × 10⁻⁸ K₁) ++++ Greaterthan wild-type activity

Example 8 GDF-11 and Activin A Binding

Binding of certain ActRIIB-Fc proteins to ligands was tested in aBiaCore™ assay.

The ActRIIB-Fc variants or wild-type protein were captured onto thesystem using an anti-hFc antibody. Ligands were injected and flowed overthe captured receptor proteins. Results are summarized in tables below.

Ligand Binding Specificity IIb Variants.

GDF11 Kon Koff KD Protein (1/Ms) (1/s) (M) ActRIIB-hFc (R64 20-134)1.34e−6 1.13e−4 8.42e−11 ActRIIB-hFc (R64, A24N 20-134) 1.21e−6 6.35e−55.19e−11 ActRIIB-hFc (R64, L79D 20-134) 6.7e−5 4.39e−4 6.55e−10ActRIIB-hFc (R64, L79E 20-134) 3.8e−5 2.74e−4 7.16e−10 ActRIIB-hFc (R64K20-134) 6.77e−5 2.41e−5 3.56e−11 GDF8 Kon Koff KD Protein (1/Ms) (1/s)(M) ActRIIB-hFc (R64 20-134) 3.69e−5 3.45e−5 9.35e−11 ActRIIB-hFc (R64,A24N 20-134) ActRIIB-hFc (R64, L79D 20-134) 3.85e−5 8.3e−4  2.15e−9ActRIIB-hFc (R64, L79E 20-134) 3.74e−5 9e−4   2.41e−9 ActRIIB-hFc (R64K20-134) 2.25e−5 4.71e−5 2.1e−10 ActRIIB-hFc (R64K 20-129) 9.74e−42.09e−4 2.15e−9 ActRIIB-hFc (R64, P129S, 1.08e−5 1.8e−4  1.67e−9 P130R20-134) ActRIIB-hFc (R64, K74A 20-134) 2.8e−5 2.03e−5 7.18e−11 ActivinAKon Koff KD Protein (1/Ms) (1/s) (M) ActRIIB-hFc (R64 20-134) 5.94e61.59e−4 2.68e−11 ActRIIB-hFc (R64, A24N 20-134) 3.34e6 3.46e−4 1.04e−10ActRIIB-hFc (R64, L79D 20-134) Low binding ActRIIB-hFc (R64, L79E20-134) Low binding ActRIIB-hFc (R64K 20-134) 6.82e6 3.25e−4 4.76e−11ActRIIB-hFc (R64K 20-129) 7.46e6 6.28e−4 8.41e−11 ActRIIB-hFc (R64,P129S, 5.02e6 4.17e−4 8.31e−11 P130R 20-134)

Other variants have been generated and tested, as reported inWO2006/012627, using ligands coupled to the device and flowing receptorover the coupled ligands. A table of data with respect to these variantsis reproduced below:

Soluble ActRIIB-Fc Variants Binding to GDF11 and Activin A (BiacoreAssay)

ActRIIB ActA GDF11 WT (64A) KD = 1.8e−7M  KD = 2.6e−7M (+) (+) WT (64R)na KD = 8.6e−8M (+++) +15tail KD ~2.6e−8M KD = 1.9e−8M (+++) (++++)E37A * * R40A − − D54A − * K55A ++ * R56A * * K74A KD = 4.35e−9 M KD =5.3e−9M +++++ +++++ K74Y * −− K74F * −− K74I * −− W78A * * L79A + *D80K * * D80R * * D80A * * D80F * * D80G * * D80M * * D80N * * D80I * −−F82A ++ − * No observed binding −− <⅕ WT binding − ~½ WT binding + WT ++<2x increased binding +++ ~5x increased binding ++++ ~10x increasedbinding +++++ ~40x increased binding

Example 9 Effect of ActRIIB-Fc on Bone Loss and Adiposity Caused byOrchidectomy

Androgen-deprivation therapy, most prominently used in the treatment ofprostate cancer, can cause pathological loss of muscle and bone, as wellas enlargement of adipose tissue. Applicants investigated effects ofActRIIB-Fc in the orchidectomized (ORX) mouse, an animal model whichmimics many of the changes associated with androgen deprivation.Nine-week-old C57BL/6 mice were ORX or sham-operated, and ten days latertreatment was initiated with ActRIIB(R64 20-134)-mFc orTris-buffered-saline (TBS) vehicle (n=10 per group) twice per week at 10mg/kg, i.p., for a period of 10 weeks (71 days).

In this experiment, ActRIIB-mFc treatment increased body weight as thenet effect of beneficial changes in muscle mass, bone mass, and fatmass. As shown in FIG. 9, ActRIIB-mFc increased the rate of body weightgain, compared to controls, under ORX conditions as well as gonad-intactconditions. This effect was due to a pronounced increase in lean bodymass. Whereas ORX controls showed a slight decline in lean body massover 10 weeks, ORX mice treated with ActRIIB-mFc displayed a markedincrease in lean body mass, reaching a mean value 25% higher thancontrols at study completion (FIG. 10). A similar increase was observedunder gonad-intact conditions for ActRIIB-mFc compared to vehicle (FIG.10). Part of this increase in lean body mass was due to a stimulatoryeffect of ActRIIB-mFc on muscle mass under both ORX conditions andgonad-intact conditions, as exemplified by three different skeletalmuscles (FIG. 11).

ActRIIB-mFc exerted a series of beneficial effects on bone. Asdetermined by whole-body analysis with dual energy X-ray absorptiometry(DEXA), ActRIIB-mFc prevented progressive decreases in bone area andbone mineral content evident under ORX conditions and led tosignificantly increased bone area and bone mineral content undergonad-intact conditions (FIGS. 12, 13). ActRIIB-mFc also increasedwhole-body bone mineral density under ORX conditions (FIG. 14).Moreover, micro-CT analysis of trabecular bone in the proximal tibiarevealed that ActRIIB-mFc treatment restored several bone parameters inORX mice to levels observed in gonad-intact controls. With respect toORX controls, these changes included: 1) a tripling of the bone volumefraction (FIG. 15), 2) a doubling of trabecular number (FIG. 16), 3)increased trabecular thickness (FIG. 17), and 4) reduced trabecularseparation (FIG. 18). The similarity of tibial morphology in ORX micetreated with ActRIIB-mFc to that in gonad-intact controls is evidentfrom images shown in FIG. 19. For each of the foregoing tibia-basedendpoints, ActRIIB-mFc also produced changes in gonad-intact micecomparable in direction and magnitude to those in ORX mice (FIGS.15-18).

ActRIIB-mFc also exerted beneficial effects on fat mass. As determinedby NMR, total fat mass in ORX controls tripled over the course of thestudy. ActRIIB-mFc treatment in ORX mice cut this increase by more than60%, restoring fat mass under ORX conditions to levels observed ingonad-intact controls (FIG. 20). ActRIIB-mFc also reduced the gain infat mass observed in gonad-intact mice during the study. Consistent withthese results, a histologic survey of fat depots indicated thatActRIIB-mFc reduced adipocyte size in subcutaneous and epididymal depotsbut not appreciably in interscapular brown fat (FIG. 21).

Finally, ActRIIB-mFc treatment altered circulating concentrations ofadiponectin and leptin, endocrine molecules originating in adiposetissue (adipokines). There is general agreement that adiponectin is akey biomarker of body composition, as circulating adiponectin levels areknown to vary inversely with fat mass/obesity, and adiponectin enhancesinsulin sensitivity in target tissues. Moreover, low adiponectin levelsare associated with cardiovascular risk factors even in nonobese healthyindividuals (Im et al., 2006, Metabolism 55:1546-1550). Thus, it isimportant that ActRIIB-mFc treatment increased serum adiponectinconcentrations significantly in both ORX and gonad-intact mice comparedto their vehicle-treated counterparts (FIG. 22). The higher adiponectinconcentrations in ORX mice compared to their gonad-intact counterpartsare consistent with the known inhibitory effect of androgen onadiponectin (Nishizawa et al., 2002, Diabetes 51:2734-2741). ActRIIB-mFcalso reduced serum concentrations of leptin, another indicator ofadipocyte status, in both ORX and gonad-intact mice compared to vehicle(FIG. 23).

Taken together, these data indicate that soluble ActRIIB-Fc chimeras canbe used as antagonists of signaling by TGF-β family ligands in males totreat bone loss and increased adiposity arising from androgendeprivation and potentially other conditions as well.

Example 10 Effect of ActRIIB-Fc Variants on Adiponectin Levels in MiceFed a High-Fat Diet

Applicants investigated the effects of ActRIIB(20-134)-hFc orActRIIB(25-131)-hFc on circulating concentrations of adiponectin in malemice fed a high-fat diet. Ten-week-old C57BL/6 mice were weight-matchedand treated subcutaneously with ActRIIB(20-134)-hFc (10 mg/kg),ActRIIB(25-131)-hFc (10 mg/kg), or Tris-buffered-saline (TBS) vehicletwice per week for 60 days. During this period, mice had unlimitedaccess to a diet containing 58% fat instead of the standard chowcontaining 4.5% fat. An additional group of mice maintained on thestandard chow diet was also treated with TBS vehicle and followed as adietary control. By Day 60, ActRIIB(20-134)-hFc treatment increasedserum adiponectin concentrations in mice fed the high-fat diet toapproximately the same levels seen in mice fed the standard diet, whileActRIIB(25-131)-hFc treatment raised serum adiponectin concentrationssignificantly beyond these control levels (FIG. 24). Contributing toelevated adiponectin concentrations was an increase in adiponectin geneexpression in white fat. Analysis of white adipose tissue by real-timepolymerase chain reaction (RT-PCR) revealed that ActRIIB(25-131)-hFcincreased adiponectin mRNA levels by more than 60% compared to high-fatdiet controls (FIG. 25).

Taken together, these findings demonstrate that ActRIIB-Fc proteins canbe used in vivo to increase adiponectin gene expression in white adiposetissue and to increase circulating adioponectin levels under a varietyof physiological conditions.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A method for increasing adiponectin in a patient in need thereof, themethod comprising administering an effective amount of a compoundselected from the group consisting of: a. a polypeptide comprising anamino acid sequence that is at least 90% identical to SEQ ID NO: 26; andb. a polypeptide encoded by a nucleic acid that hybridizes understringent hybridization conditions to the nucleic acid of SEQ ID NO: 3.2. The method of claim 1, wherein the polypeptide is a fusion proteincomprising a portion heterologous to ActRIIB.
 3. The method of claim 1,wherein the polypeptide is a dimer.
 4. The method of claim 2, whereinthe polypeptide is fused to a constant domain of an immunoglobulin. 5.The method of claim 2, wherein the polypeptide is fused to an Fc portionof an immunoglobulin.
 6. The method of claim 5, wherein theimmunoglobulin is a human IgG1.
 7. The method of claim 1, wherein thepatient has adiponectin deficiency or insufficiency.
 8. The method ofclaim 1, wherein the patient has low circulating concentrations ofadiponectin.
 9. The method of claim 1, wherein the polypeptide comprisesan amino acid sequence that is at least 95% identical to SEQ ID NO: 26.10. The method of claim 1, wherein the polypeptide comprises an aminoacid sequence that is at least 97% identical to SEQ ID NO:
 26. 11. Themethod of claim 1, wherein the polypeptide comprises an amino acidsequence that is at least 99% identical to SEQ ID NO:
 26. 12. The methodof claim 1, wherein the polypeptide comprises an amino acid sequencethat is at least 95% identical to SEQ ID NO:
 5. 13. The method of claim1, wherein the polypeptide comprises an amino acid sequence that is atleast 97% identical to SEQ ID NO:
 5. 14. The method of claim 1, whereinthe polypeptide comprises the amino acid sequence of SEQ ID NO:
 5. 15.The method of claim 1, wherein the polypeptide comprises an amino acidsequence that is at least 95% identical to SEQ ID NO:
 23. 16. The methodof claim 1, wherein the polypeptide comprises an amino acid sequencethat is at least 97% identical to SEQ ID NO:
 23. 17. The method of claim1, wherein the polypeptide comprises the amino acid sequence of SEQ IDNO: 23
 18. The method of claim 1, wherein administration of the compoundincreases adiponectin expression in adipocytes of the treated patient.19. A method for increasing adiponectin in a patient in need thereof,the method comprising administering an effective amount of a compoundselected from the group consisting of: a. an antagonist of ActRIIB; b.an antagonist of myostatin; c. an antagonist of BMP7; d. an antagonistof Activin A and/or B; and e. an antagonist of GDF3.
 20. The method ofclaim 19, wherein the compound is an antagonist of ActRIIB.
 21. Themethod of claim 20, wherein the antagonist of ActRIIB is selected fromthe group consisting of: an antibody that binds to ActRIIB and a nucleicacid that hybridizes to a nucleic acid encoding ActRIIB and inhibitsActRIIB production.
 22. The method of claim 19, wherein the compound isan antagonist of myostatin.
 23. The method of claim 20, wherein theantagonist of myostatin is selected from the group consisting of: anantibody that binds to myostatin, a nucleic acid that hybridizes to anucleic acid encoding myostatin and inhibits myostatin production, and apolypeptide comprising a myostatin propeptide or variant thereof. 24.The method of claim 19, wherein the compound is an antagonist of BMP7.25. The method of claim 20, wherein the antagonist of BMP7 is selectedfrom the group consisting of: an antibody that binds to BMP7 and anucleic acid that hybridizes to a nucleic acid encoding BMP7 andinhibits BMP7 production.
 26. The method of claim 19, wherein thecompound is an antagonist of Activin A and/or Activin B.
 27. The methodof claim 26, wherein the antagonist of Activin A and/or Activin B isselected from the group consisting of: a. an antibody that binds toActivin A; b. an antibody that binds to Activin B; c. an antibody thatbinds both Activin A and Activin B; d. a nucleic acid that hybridizes toa nucleic acid encoding Activin A and inhibits Activin A production; e.a nucleic acid that hybridizes to a nucleic acid encoding Activin B andinhibits Activin B production; and f. a nucleic acid that hybridizes toa nucleic acid encoding Activin A and a nucleic acid encoding Activin Band inhibits both Activin A and Activin B production.
 28. The method ofclaim 19, wherein the compound is an antagonist of GDF3.
 29. The methodof claim 28, wherein the antagonist of GDF3 is selected from the groupconsisting of: an antibody that binds to GDF3, a nucleic acid thathybridizes to a nucleic acid encoding GDF3 and inhibits GDF3 production,and a polypeptide comprising a GDF3 propeptide or variant thereof.
 30. Amethod for ameliorating one or more undesired effects of anti-androgentherapy in a patient in need thereof, the method comprisingadministering to the patient an effective amount of an ActRIIB fusionprotein, wherein the ActRIIB fusion protein comprises an amino acidsequence that is at least 95% identical to the sequence corresponding toamino acids 29-109 of SEQ ID NO:
 2. 31. The method of claim 30, whereinthe ActRIIB fusion protein comprises a portion derived from the ActRIIBsequence of SEQ ID NO: 2 and a second polypeptide portion, wherein theportion derived from SEQ ID NO: 2 corresponds to the sequence beginningat any of amino acid 22-25 of SEQ ID NO: 2 and ending at any of aminoacids 133-134 of SEQ ID NO: 2, and wherein the portion derived from SEQID NO: 2 differs at no more than five amino acid positions from thecorresponding sequence of SEQ ID NO:
 2. 32. The method of claim 30,wherein the ActRIIB fusion protein comprises a portion derived from theActRIIB sequence of SEQ ID NO: 2 and a second polypeptide portion,wherein the portion derived from SEQ ID NO: 2 corresponds to thesequence beginning at amino acid 25 of SEQ ID NO: 2 and ending aminoacid 131 of SEQ ID NO:
 2. 33. The method of claim 30, wherein theActRIIB fusion protein comprises a portion derived from the ActRIIBsequence of SEQ ID NO: 2 and a second polypeptide portion, wherein theportion derived from SEQ ID NO: 2 corresponds to the sequence beginningat amino acid 20 of SEQ ID NO: 2 and ending at amino acid 134 of SEQ IDNO:
 2. 34. The method of any of claims 30-33, wherein the undesiredeffect of anti-androgen therapy is muscle loss.
 35. The method of any ofclaims 30-33, wherein the undesired effect of anti-androgen therapy isbone loss.
 36. The method of any of claims 30-33, wherein the undesiredeffect of anti-androgen therapy is increased adiposity.
 37. The methodof any of claims 30-33, wherein the undesired effect of anti-androgentherapy is increased insulin resistance.
 38. The method of any of claims30-33, wherein the undesired effect of anti-androgen therapy is acombination of three or more of the following: muscle loss, bone loss,increased adiposity and insulin resistance.
 39. A method for increasingmuscle, increasing bone, and decreasing fat in a patient in needthereof, the method comprising administering to the patient an effectiveamount of an ActRIIB fusion protein, wherein the ActRIIB fusion proteincomprises an amino acid sequence that is at least 95% identical to SEQID NO:
 26. 40. The method of claim 37, wherein the patient is in need ofrelief from one or more undesired effects of androgen-deprivationtherapy